Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths due to its late diagnosis, limited treatment options, and early metastasis, resulting in a five-year survival rate of less than 10 %. Central to PDAC development are mutations in the KRAS gene, which drive tumor growth through persistent activation of signaling pathways. The microbiota, comprising bacteria, viruses, and fungi, plays a vital role in human health by aiding digestion, supporting immune function, and maintaining microbial balance. Dysbiosis, an imbalance in microbial communities, is linked to various diseases. Recent research highlights the complex interaction between the pancreas and gut microbiota, with evidence suggesting that microbiota influences pancreatic functions, including enzyme secretion and glucose regulation. This review focuses on the role of microbiome dysbiosis in PDAC, influencing tumorigenesis, immune response, and treatment outcomes through complex interactions with genetic mutations and metabolic pathways, and on the role of the microbiota in influencing KRAS-driven tumorigenesis, where dysbiosis may promote tumor progression. This review also aims to emphasize how the gut microbiome has been implicated in modulating the response to immune checkpoint inhibitors (ICIs) across various cancers, with specific bacterial strains linked to improved outcomes. Recent studies suggest that microbial manipulation, such as bacterial ablation, can enhance anti-tumor immunity in PDAC models by altering the tumor microenvironment (TME). The TME remains a major barrier, and ongoing research is exploring new combinations and targets to overcome resistance and improve the efficacy of ICIs in PDAC. Further research is needed to fully understand this relationship.

Liver cancer is a global health challenge and its incidence is expected to grow worldwide to 1 million new cases in 2025. While hepatocellular carcinoma (HCC) is the most common liver cancer, hepatic metastases of colorectal cancer (CRLM) occur in almost 50% of patients with CRC. For both HCC and CRLM, surgical resection represents the only chance of long-term survival. In this context, a label-free optical surgical guidance tool would be highly suitable for reducing the positive margin rate and improving patient outcomes. In this study, we used a fiber-based autofluorescence lifetime imaging probe to provide real-time discrimination of tumor from marginal tissues in freshly excised liver samples. The proposed method allowed discriminating tumors from healthy tissue by reporting the fluorescence lifetime decay of cellular metabolic biomarkers, i.e., NAD(P)H and FAD. The acquisitions on 37 surgical specimens of both HCC and CRLM demonstrate that this approach is a method for delineating tumor borders with 67.7% and 77% accuracy, respectively. The results, combined with the ability to capture and process images in real time under bright background, highlight the technology's potential for clinical use for both label-free tissue diagnostics and surgical guidance.

In this study, we propose a novel fibre-based autofluorescence lifetime imaging probe for the diagnosis of hepatic cancer. Our approach facilitates real-time tumour margin delineation, thereby providing metabolic insights that enhance diagnostics and surgical precision. (c) 2025 The Author(s)

We present a fiber-based autofluorescence lifetime imaging probe for hepatic cancer diagnostics. Our approach enables real-time tumor margin delineation and differentiation between different tumors, providing metabolic insights that enhance oncological diagnostics and surgical precision. (c) 2025 The Author(s)

BACKGROUND:Minimally invasive distal pancreatectomy offers recognised benefits over open surgery. Robotic surgery, with its shorter learning curve and technical advancements, presents a promising alternative to laparoscopy in managing pancreatic diseases. METHODS:This study enrolled consecutive patients undergoing distal pancreatectomy between January 2013 and May 2022. After propensity score matching, perioperative and medium-term outcomes were compared between robotic and open procedures. RESULTS:Among 79 patients analysed, 50 were eligible after matching. Both groups showed similar demographics, with 11% experiencing grade III-IV complications, and one patient died within 90 days after surgery. Robotic surgery exhibited longer operating times but allowed for earlier refeeding, drain removal, canalisation, and shorter hospital stays compared with open surgery. Spleen preservation rates, postoperative pancreatic fistula occurrences and survival were comparable between the groups. CONCLUSIONS:Overall, robotic and open distal pancreatectomy demonstrated similar outcomes, with robotic surgery offering advantages in certain postoperative parameters despite longer operation times.

Indocyanine green (ICG) is an inert polypeptide that almost totally binds to high molecular weight plasma proteins; it is cleared by the hepatocytes and directly excreted into the bile with a half-life of about 3-5 minutes. Specific systems are required to see fluorescent images. The use of this dye has been reported in different surgical specialties, and the applications in hepatobiliary surgery are widening. Being firstly used to evaluate the preoperative liver function, intra- and postoperative dynamic checking of hepatic activity has been reported and integrated within perioperative protocols allowing a tailored treatment allocation. Intravenous injection (IV) or injection into the gallbladder can ease difficult cholecystectomy. Biliary leakage detection could be enhanced by IV ICG injection. Although with some contrasting results, the use of ICG for both delineating the limits of the resection and tumor-enhanced visualization was demonstrated to improve short- and long-term outcomes. Although the lack of strong evidence still precludes the introduction of this tool in clinical practice, it harbors great potential in liver surgery.

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex-vivo. We report the application of an instrument employing an optical fiber probe and capable of performing real-time autofluorescence lifetime imaging at a macroscopic scale, under bright background conditions. We validate and demonstrate the practicality of this technology to discriminate healthy against neoplastic tissue in freshly excised tumor biopsies. The capability of delineating tumor margins through processing the fluorescence decays in the phasors domain was demonstrated on four different types of cancer, highlighting the broad range of potential clinical applications for the proposed approach. The presented results suggest that our autofluorescence lifetime imaging probe, together with phasor analysis, can offer a real-time tool to observe lifetime contrast on tissues and, thus, is a suitable candidate for improving in situ tissue diagnostics during surgery.

We demonstrate the capability of a time-resolved autofluorescence lifetime imaging setup for discriminating, perilesional and tumor tissues in freshly excised liver samples. In particular, freshly excised liver biopsies were collected from the surgery room and imaged within 30 minutes using 445 nm as excitation wavelength and one spectral window for detection. Differences in mean fluorescence lifetime were observed among the examined tissue types, potentially allowing their discrimination and classification. Interestingly, the obtained autofluorescence lifetime values were significantly different when comparing primary tumors of liver with colorectal tumor metastasis to the liver, underlying the classification capability of this experimental setup. The presented approach offers real-time acquisition and processing, optical flexibility, as well as the possibility to acquire autofluorescence data under bright background conditions. Hence, it meets all the requirements for label-free diagnostics and surgical guidance in various clinical and histopathological applications.

Background The immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that (i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and (ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases. Methods The expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of hypoxia-inducible factor (HIF)-1α on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses. Results We show that Rai is a novel HIF-1α-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted Programmed cell Death protein (PD)-1 expression and impaired antigen-dependent degranulation of CD8 + T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD-1 expression and T cell-mediated anti-tumor immunity. Conclusions Our data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells.

e23510 Background: Lynch Syndrome due to mutations in one of DNA mismatch repair (MMR) genes, is found in patients with colon cancer (3%).The mutations in these DNA repair genes induce genetic instability and development of ovarian, endometrial, urothelial, gastrointestinal and biliary tract carcinoma. Methods: In our center, 31 patients with Lynch syndrome are followed in Oncology Department in multidisciplinary team according to regional diagnostic therapeutic assistance paths and international guidelines. Immunohistochemical staining (IC) for mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) and microsatellite instability testing (MSI) by PCR real time easyPGX were performed in colon cancer, genetic testing has been preceded by genetic counseling. When GIST diagnosis occurred in patients with colon cancers and Lynch syndrome, IC and MSI were performed in GIST too. Results: MSI-H has been detected in synchronous colo-rectal cancer in 2 patients with GIST. Pathogenetic germline mutations in MSH2 gene were observed in both patients. In 1 female patient with gist of duodenum (very low risk according to Miettinen classification) with previous endometrial cancer and sincronous PT3N0 rectal cancer, we observed MSI-stable in IC and PCR, KIT/PDGFRα wild type in Next GenSequencing analysis, BRAF wild type (PCR-real time), SDH-B + (IC), NTRK 1 (exon 9-10, exon 1-12 del, exon 12), NTRK 2 (exon 12-15.exon 16-17), exon 14, NTRK 3 (exon 14, exon 15) no gene fusion by PCR-real time (CE.IVD kit easy NTRK). In 1 male patient with ileal GIST (intermediate risk according to Miettinen classification ) with ileal adenocarcinoma pT3N0, we observed KIT exon 11 mutated, MSI-stable in IC, when we repeated the analysis using PCR real time EasyPGX, we observed high instability of microsatellites. Conclusions: For the first time we observed in Lynch Syndrome MSH2 mutated, simolutaneous GIST and colon cancer in the same site of ileum and PCR analisys detected a rare exemple of microsatellite instability in GIST.

Esophageal pressure, bile content and pH are important parameters in gastroesophageal diseases. An all-optical technology is described to perform simultaneously oesophageal manometry, pH-metry and bilimetry. The pressure measurement along the oesophagus is performed with 11 fibre optic gratings (FBGs) within a single mode fibre, which ensures monitoring every 2.5 cm along the oesophagus. The bile measurement is based on the direct absorption of bilirubin, the main biliary pigment, using a bundle of plastic optical fibres (POFs) carrying the signal. Regarding the detection of pH, an acid-base indicator that modifies its absorption as a function of pH is immobilized on glass particles with controlled porosity immobilized at the end of two POFs. The sensors are integrated in the same catheter for the simultaneous measurement of the three parameters. The prototype for the catheter interrogation was designed and developed, constituted by the integration of two optoelectronic modules for the interrogation of the pressure sensor and for the bile and pH sensors, respectively.

Pancreatic ductal adenocarcinoma (PDAC) is one of the big killers with a 5-year-survival rate of less than 2% in metastatic disease. Limited treatment options and early clinical deterioration often affect outcome. Exocrine pancreatic insufficiency (EPI) is often underestimated in patients with advanced PDAC and a large proportion of patients (60-80%) does not receive pancreatic enzyme replacement therapy (PERT). This study aims to optimize the clinical selection of patients with advanced PDAC treated with gemcitabine/nabpaclitaxel and to develop models to predict benefit from first-line therapy and duration of second-line therapy. In this observational study we prospectively enrolled patients with advanced PDAC treated with gemcitabine/nabpaclitaxel as first-line therapy at the Medical Oncology Unit of Careggi University Hospital from 2015 to 2022. Clinical and laboratory data were collected and associated with survival outcomes, duration of second-line therapy and treatment exposure. A total of 107 patients were enrolled, 54 (50.5%) were women and 53 (49.5%) were men. Disease stage was classified as locally advanced in 42 (39.3%) and metastatic in 65 (60.7%). Median progression-free survival (PFS) was 5.6 months and median overall survival (OS) was 7.4 months. Among the baseline clinical variables, a trend towards a worse prognosis was observed in patients with a high tumor burden (OS p=0.065). Overall, 66.0% of patients received PERT at baseline or within 3 months from first-line treatment start. PERT-users had a significantly longer OS than non-PERT-users (9.5 months vs 5.5 months, respectively, HR 95%CI 2,1 [1,2-3,7], p=0.008). In addition, the probability to receive a second-line treatment for at least 2 months was higher in the PERT-user group than in the non-PERT-user group (X-squared=7.6558, p-value=0.005659). The clinical management of patients with advanced PDAC is challenging and requires a multidisciplinary strategy to prevent EPI-related symptoms and optimize treatment adherence. PERT could play a crucial role in preventing weight loss, maintaining dose intensity and improving survival.

IntroductionPancreatic adenocarcinoma (PC) is one of the most lethal malignancies; even after resection the patients’ 5-year disease-free survival (DFS) is lower than 26%. The genetic mutational landscape of PC is dominated by activating KRAS mutations, that have been reported in approximately 90% of cases; however, beyond KRAS - direct mutations, several KRAS-targeting miRNAs appear to be downregulated, strengthening the already activated RAS signaling. In addition, the interplay between miRNAs and RAS includes poorly investigated downstream miRNAs. The aim of this study was to determine the prognostic value of some of these candidate KRAS-related miRNAs.Patients and methodsBetween 2015 and 2022, 44 patients with pathologically confirmed PC, who received surgery and were enrolled by the Clinical Oncology Unit, Careggi University Hospital, Florence (Italy). PC Total RNA was extracted from FFPE sections, retro-transcribed and the resulting cDNA was then used for qPCR analysis. A panel of KRAS-related miRNA (miR-155, miR-206 and miR-143) was analyzed.ResultsIn this observational study patients sex distribution was unequal with 34.1% being male and 65.9% female. The most frequent tumor localization was the head of the pancreas (65.9%) and the pathological stages were pT1-2 (45.5%), pT3 (54.5%), pN0 (22.7%), pN+ (77.3%). Adjuvant therapy was administered to 63.6% of patients; disease recurrence was observed in 69% of cases. Twenty-three patients, whose RNA was of adequate quality, were used in the mRNAs expression studies. When comparing the miRNA expression between PC and a pool of healthy tissues, miR-155 was overexpressed and miR-206 downregulated in PC, while miR-143 expression was unchanged. However, when categorized in low- and high- miR-143 expressing PC (according to the median value), high miR-143 was associated with nodal involvement (pN+) (p=0.029), who in turn was linked with shorter DFS (p=0.009) and overall survival (OS) (p=0.021) compared to pN0. A trend toward inferior DFS was observed for higher expression of miR-206 (p=0.095) and miR-143 (p=0.092). Finally, responders to a first-line treatment for advanced disease had miR-155 overexpressed (p=0.048).ConclusionsmiRNAs are involved in PC tumorigenesis and metastatic spread. In light of miR-143 association with lymphatic spread and poor prognosis, a comprehensive analysis of miRNA interplay with KRAS deserves further investigation.

e16314 Background: Pancreatic adenocarcinoma (PC) is one of the big killers with a 5-year disease-free survival (DFS) rate of 19-26% after resection despite adjuvant treatments. KRAS mutations have been reported in approximately 90% of PC cases and, beyond KRAS activation by gene mutations, several miRNAs directly targeting KRAS have been reported to be simultaneously downregulated, strengthening the already activated RAS signaling. In addition, the interplay between miRNAs and RAS includes downstream miRNA effectors whose role needs to be elucidated. The aim of this study was to evaluate the expression of candidate miRNAs strictly related to KRAS expression in order to elucidate the prognostic value. Methods: In this single-center observational prospective study we included patients with pathologically confirmed PC, who underwent radical surgery and were referred to the Clinical Oncology Unit, Careggi University Hospital, Florence (Italy), between 2015 and 2022. Total RNA was extracted from FFPE sections, retro-transcribed and the resulting cDNA was then used for qPCR analysis. Results: Among 44 patients included in the study, 34.1% were male and 65.9% female (median age 75 years). The majority of cases had the primary tumor located at the head of the pancreas (65.9%) and the pathological stage was pT1-2 in 45.5%, pT3 in 54.5%, pN0 in 22.7%, pN+ in 77.3%. Overall, 63.6% of patients received adjuvant chemotherapy and 69.0% experienced disease recurrence. Twenty-three specimens were adequate for miRNA analysis. A different expression of the three target miRNAs between PC and healthy tissue was observed: miR-155 was overexpressed and miR-206 downregulated in PC, while miR-143 had a similar expression in both tissues. miR-143 overexpression was associated with nodal involvement (pN+) at the pathological examination (p = 0.029). Among clinicopathological features, pN+ was associated with shorter DFS (p = 0.009) and overall survival (OS) (p = 0.021) compared to pN0. A trend toward inferior DFS was observed for higher expression of miR-206 (p = 0.095) and miR-143 (p = 0.092). Among patients receiving a first-line treatment for advanced disease, miR-155 was overexpressed in responders vs non-responders (p = 0.048). Conclusions: miRNA expression is involved in tumorigenesis and metastatic spread in PC. A comprehensive analysis of the miRNA-RAS interplay deserves to be further investigated as a predictor of clinical outcome, especially for miR-143 overexpression which was associated with lymphatic spread and, consequently, poor prognosis.

Esophageal pressure, bile content and pH are important parameters in gastroesophageal diseases. An all-optical technology is described capable to perform simultaneously oesophageal manometry, pH-metry and bilimetry. The three different sensors were integrated in a single optical fibre catheter for the simultaneous measurement of the three parameters. The optoelectronic prototype for the interrogation of the catheter is constituted by two separate optoelectronic modules for the interrogation of the pressure sensor and for the bile and pH sensors, respectively. The prototype and the optical catheter are compliant with European Directives on medical devices in terms of electromagnetic compatibility, electrical safety and biocompatibility.

Background: This study assessed the potential cost-effectiveness of high (80–100%) vs low (21–35%) fraction of inspired oxygen (FiO2) at preventing surgical site infections (SSIs) after abdominal surgery in Nigeria, India, and South Africa. Methods: Decision-analytic models were constructed using best available evidence sourced from unbundled data of an ongoing pilot trial assessing the effectiveness of high FiO2, published literature, and a cost survey in Nigeria, India, and South Africa. Effectiveness was measured as percentage of SSIs at 30 days after surgery, a healthcare perspective was adopted, and costs were reported in US dollars ($). Results: High FiO2 may be cost-effective (cheaper and effective). In Nigeria, the average cost for high FiO2 was $216 compared with $222 for low FiO2 leading to a −$6 (95% confidence interval [CI]: −$13 to −$1) difference in costs. In India, the average cost for high FiO2 was $184 compared with $195 for low FiO2 leading to a −$11 (95% CI: −$15 to −$6) difference in costs. In South Africa, the average cost for high FiO2 was $1164 compared with $1257 for low FiO2 leading to a −$93 (95% CI: −$132 to −$65) difference in costs. The high FiO2 arm had few SSIs, 7.33% compared with 8.38% for low FiO2, leading to a −1.05 (95% CI: −1.14 to −0.90) percentage point reduction in SSIs. Conclusion: High FiO2 could be cost-effective at preventing SSIs in the three countries but further data from large clinical trials are required to confirm this.

Background: Approximately 95% of Colorectal cancers (CRC) consist of adenocarcinomas originating from colonic Adenomatous polyps (AP). Increasing importance in CRC occurrence and progression has been attributed to the gut microbiota; however, a huge proportion of microorganisms inhabit the human digestive system. So, to comprehensively study the microbial spatial variations and their role in CRC progression, from AP to the different CRC phases, a holistic vision is imperative, including the simultaneous evaluation of multiple niches from the gastrointestinal system. Through an integrated approach, we identified potential microbial and metabolic biomarkers, able to discriminate human CRC from AP and/or also the different Tumor node metastasis (TNM) staging. In addition, as the microbiota contributes to the production of essential metabolic products detectable in fecal samples, we analysed and compared metabolites obtained from CRC and AP patients by using a Nuclear magnetic resonance (NMR) approach. Methods: In this observational study, saliva, tissue and stool samples from 61 patients, have been collected, including 46 CRC and 15 AP patients, age and sex-matched, undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). First, the microbiota in the three-district between CRC and AP patients has been characterized, as well as in different CRC TNM stages. Subsequently, proton NMR spectroscopy has been used in combination with multivariate and univariate statistical approaches, to define the fecal metabolic profile of a restricted group of CRC and AP patients. Results: CRC patients display a different profile of tissue and fecal microbiota with respect to AP patients. Significant differences have been observed in CRC tissue microbial clades, with a rise of the Fusobacterium genus. In addition, significant taxa increase at the genus level has been observed in stool samples of CRC patients. Furthermore, Fusobacterium found in intestinal tissue has been positively correlated with fecal Parvimonas, for the first time. Moreover, as predicted by metagenomics pathway analysis, a significant increase of lactate (p=0.037) has been observed in the CRC fecal metabolic profiles, and positively correlated with Bifidobacterium (p=0.036). Finally, minor bacterial differences in CRC patients at stage T2 (TNM classification) have been detected, with a raise of the Spirochaetota phylum in CRC samples, with a slight increase of the Alphaproteobacteria class in fecal samples. Conclusion: Our results suggest the importance of microbiota communities and oncometabolites in CRC development. Further studies on CRC/AP management with a focus on CRC assessment are needed to investigate novel microbial-related diagnostic tools aimed to improve therapeutic interventions.